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論文中文名稱:源自於腸道之親蛋白尿毒素對於血管內皮前驅細胞功能與慢性腎病變患者臨床預後之研究 [以論文名稱查詢館藏系統]
論文英文名稱:The effects of GI-related protein-bound uremic toxins on endothelial progenitor cell function and clinical outcomes in chronic kidney disease [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:工程科技研究所
畢業學年度:102
出版年度:103
中文姓名:林承叡
英文姓名:Cheng-Jui Lin
研究生學號:100679016
學位類別:博士
語文別:中文
口試日期:2014-06-05
論文頁數:54
指導教授中文名:劉宣良
口試委員中文名:吳志仁;莊志光;黃志宏;李勝祥
中文關鍵詞:血管內皮細胞血管內皮前趨細胞慢性腎病變親蛋白尿毒素對硫甲酚硫酸吲哚酚血液透析腹膜透析心血管疾病週邊動脈阻塞血管通路失敗
英文關鍵詞:Endothelial cellEndothelial progenitor cellChronic kidney diseaseProtein-bound uremic toxinP-cresyl sulfateIndoxyl sulfateHemodialysisPeritoneal dialysisCardiovascular diseasePeripheral artery diseaseVascular access failure
論文中文摘要:心血管疾病(cardiovascular disease)長期以來一直都是導致慢性腎臟病(chronic kidney disease, CKD)患者死亡之最主要原因。然而,傳統的心血管危險因素(traditional risk factors)不能完全解釋CKD患者心血管疾病之風險。最近的研究顯示非傳統的心血管風險因素(non-traditional risk factors),其中包括親蛋白尿毒素(protein-bound uremic toxins)- 對硫甲酚(p-cresylsulfate)與硫酸吲哚酚(indoxyl sulfate)也可能扮演重要的角色。內皮細胞(endothelial cell)功能異常被證實是造成CKD患者心血管疾病之關鍵因素。過去體外試驗顯示,對硫甲酚與硫酸吲哚酚濃度升高會增加氧化壓力及自由基,並造成血管內皮細胞和平滑肌細胞之功能異常。而血管內皮前趨細胞(endothelial progenitor cells)則被認為可幫助內皮細胞修復及促進血管新生,對硫甲酚與硫酸吲哚酚是否會導致血管內皮前趨細胞功能異常仍不清楚。
因此本論文將從體外(in vitro)至人體(in vivo)實驗探討並驗證此二尿毒素之生理毒性。細胞實驗顯示硫酸吲哚酚對於血管內皮細胞及血管內皮前趨細胞之血管新生作用(angiogenesis)有明顯抑制作用。隨著尿毒素濃度升高, 血管內皮前趨細胞之移行(migration)、細胞菌落形成 (colony forming unit) 與細胞增生(proliferation)也明顯受到抑制。然而在臨床人體研究可發現,隨著腎臟功能衰退時,人體之血管內皮細胞功能(Flow mediate dilation)則愈差,在經過多變項迴歸分析後,其也與血液中對硫甲酚、硫酸吲哚酚濃度有高度之關聯性。
此外我們也針對多項之臨床預後指標探討此二尿毒素之臨床角色。研究顯示對於慢性腎病變第三至五期患者,對硫甲酚、硫酸吲哚酚可成功預測病患之心血管事件與腎臟功能衰退;在血液透析患者,對硫甲酚則與病患之感染住院率、心血管疾病、死亡率、週邊動脈阻塞及血管通路阻塞率有高度的正相關;對於腹膜透析患者,此二尿毒素對於評估患者之心血管疾病、死亡率及腹膜透析失敗率等,也明顯達到統計學上差異。
綜合上述由體外至人體研究之結果,證實血清中對硫甲酚、硫酸吲哚酚濃度確實與臨床預後有緊密關聯性。我們推測此二毒素可能藉由抑制血管內皮前趨細胞功能,進而影響血管內皮細胞功能之完整性,最終導致慢性腎病變患者有較差之臨床預後。
論文英文摘要:Cardiovascular diseases (CVD) are still the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Development of accelerated atherosclerosis involves multiple risk factors. However, traditional risk factors could not fully account for the high risk of CVD in CKD patients. Recent studies supported the idea of non-traditional risk factors, which included renal anemia, oxidative stress and uremic toxins. Protein-bound uremic toxins including indoxyl sulfate (IS) and p-cresyl sulfate (PCS) accumulated while renal function decline have been reported having adverse effect on endothelial cells (ECs) function by increasing production of oxidative stress and free radical. Endothelial dysfunction has been regarded to be the essential step resulting in CVD. However, recent studies also showed endothelial progenitor cells (EPCs) could function to restore the endothelial function damaged during coronary ischemia. It is still unclear whether IS or PCS will lead to EPCs dysfunction.
Thus, our purpose is to further investigate the pathological effects of IS and PCS from in vitro to in vivo study. Our results showed that IS had obvious negative effect on angiogenesis of human umbilical vein endothelial cells (HUVECs) and EPCs. The ability of EPCs migration, colony forming unit and proliferation were also inhibited by IS and PCS in a dose dependent manner. In addition, we also found the human endothelial function evaluated by flow mediate dilation (FMD) was significantly decreased in advanced CKD. The FMD value was negatively correlated to serum IS and PCS levels after adjusting other confounding factors.
Moreover, we also explore the effects of IS and PCS on multiple clinical outcomes in CKD. Form our research, it showed IS and PCS were capable of predicting CVD event and kidney function deterioration in patients with CKD stage 3-5. For hemodialysis patients, PCS was strongly associated with CVD event, hospitalization event and vascular access failure. Both toxins were also a valuable surrogate to evaluate the event of CVD, mortality and peritoneal dialysis (PD) failure in PD cohort.
From our results above-mentioned, it indicated that higher serum IS and PCS levels were closely related to worse clinical outcomes. We speculate that these adverse outcomes may contribute directly or indirectly to the loss of endothelial cell function owing to inhibition of EPCs function by IS or PCS.
論文目次:ABSTRACT………………………………………………...………………...................i
ACKNOWLEDGEMENTS….......................………………………………….............v
CONTENTS………………………………………………….......................……........vi
TABLE CONTENTS................................................................................................viii
FIGURE CONTENTS............................................................................................... ix
Chapter 1. INTRODUCTION.....................................................................................1
1.1 Cardiovascular risk of chronic kidney disease…….…..................................1
1.2 Production of IS and PCS…………………………….……..…….................1
1.3 Toxicity of IS and PCS…………………………….…………......................2
1.4 Role of endothelial progenitor cells………………….…………...................2
1.5 Aim of study…………………………………………..…………..................3
Chapter 2. MATERIALS and METHODS……………………………....…............4
2.1 Basic study………………………………………………..................…….…....4
2.1.1 Isolation and Cultivation of EPCs………………………..................…………4
2.1.2 Characterization of early EPCs…………………………..................………….4
2.1.3 EPCs Colony Forming Assay………………………….....................................5
2.1.4 EPCs Migration Assay………………………………………….......................5
2.1.5 EPCs Proliferation Assay……………………………… …..…........................6
2.1.6 Angiogenesis Assay……………………………………………..................….6
2.1.7 Statistical Analysis……………………………………………................….….6
2.2. Human study…………………………………………………................….…..6
2.2.1 Human endothelial function measurement….....................................................6
2.2.2 Measurement of Artery stiffness…………………….........................................7
2.2.3 Laboratory assessment………………................................................................7
2.2.4 IS and PCS measurement……………………………………............................7
2.2.5 Study patients………………………………………………….....................8
2.2.6 End point evaluation……………………………………………………….9
2.2.7 Statistical Analysis…………………………………………………………....10
Chapter 3. RESULT…………………………………………………….......................11
3.1 Basic study……………………………….…………………..........................11
3.1.1 Effect of IS and PCS on HUVECs.…………………………….......................11

3.1.2 Effect of IS and PCS on EPCs………………………………………......….13
3.1.2.1 Cell character and viability………………………………………………....13
3.1.2.2 Angiogenesis of EPCs……………………………………………….……..15
3.1.2.3 EPCs chemotaxis and colony forming assay……………………….............17
3.2 Human study……………………………………………………………….20
3.2.1 Endothelial function in CKD………………………………………………20
3.2.2 Clinical outcomes in CKD………………………………………………...25
3.2.3 Clinical outcome in HD patients………………………………..…..……..30
3.2.4 Clinical outcomes in PD patients………………………………….............35
3.2.5 Vascular outcomes in HD patients………………………...........................40
Chapter 4. DISCUSSION AND CONCLUSION……………………………………..45
REFERENCES………..…………………………………………………….……..….…...48
ABBREVIATION……….………..………………………………...............................54
論文參考文獻:1. M. J. Samak, A. S. Levey, A. C. Schoolwerth, et al. “Kidney disease as a risk factor for development of cardiovascular disease: A statement from the American Heart Association councils on kidney in cardiovascular disease, high blood pressure research, clinical cardiology, and epidemiology and prevention,” Hypertension, vol. 42, no. 5, 2003, pp. 1050-1065.
2. A. S. Go, G. M. Chertow, D. Fan, et al. “Chronic kidney disease and the risks of death, cardiovascular events, andhospitalization,” N Engl J Med, vol. 351, no. 13, 2004, pp. 1296-1305.
3. D. S. Keith, G. A. Nichols, C. M. Gullion, et al. ”Longitudinal follow-up and outcomes among a population with chronic kidney disease in a large managed care organization,” Arch Intern Med, vol.164, no. 6, 2004, pp. 659-663.
4. M. J. Sarnak and A. S. Levey, ” Cardiovascular disease and chronic renal disease: a new paradigm,” Am J Kidney Dis Suppl, vol. 35, no. 4, 2000, pp. S117-S131.
5. B. F. Culleton and P. W. F. Wilson, ” Cardiovascular disease: risk factors, secular trends and therapeutic guidelines,” J Am Soc Nephrol, vol. 9, no. 12 suppl, 1998, pp.5-15.
6. J. C. Longenecker , J. Coresh , N. R. Powe , et al. ”Traditional cardiovascular disease risk factors in dialysis patients compared with the general population: the CHOICE Study,” J Am Soc Nephrol, vol.13, no. 7, 2002, pp. 1918-1927.
7. R. Clarke, L. Daly, K. Robinson, et al. “Hyperhomocysteinemia: an independent risk factor for vascular disease,” N Engl J Med, vol. 324, no. 17, 1991, pp. 1149-1155.
8. B. Bammens, P. Evenepoel, H. Keuleers, et al. ”Free serum concentrations of the proteinbound retention solute p-cresol predict mortality in hemodialysis patients,” Kidney Int, vol.69, no. 6, 2006, pp.1081-1087.
9. B. K. Meijers , B. Bammens , B. De Moor , et al. “Free p-cresol is associated with cardiovascular disease in hemodialysis patients,” Kidney Int, vol. 73, no. 10, 2008, pp. 1174-1180.
10. C. J. Lin, C. J. Wu, C. F. Pan, et al. “Serum protein-bound uraemic toxins and clinical outcomes in haemodialysis patients,” Nephrol Dial Transplant, vol. 25, no. 11, 2010, pp. 3693-3700.
11. R. Ross, “Atherosclerosis-an inflammatory disease,” N Engl J Med, vol.340, no. 2, 1999, pp. 115-126.
12. D. C. Wheeler, ”Cardiovascular disease in patients with chronic renal failure,” Lancet, vol. 348, 1996, pp. 1673-1674.
13. T. Miyazaki, M. Ise, M. Hirata, et al. “Indoxyl sulfate stimulates renal synthesis of transforming growth factor-β1 and progression of renal failure,” Kidney Int Suppl, vol. 63, 1997, pp. S211-S214.
14. T. Miyazaki, M. Ise, H. Seo, et al. ”Indoxyl sulfate increases the gene expressions of TGF-b1, TIMP-1 and proa1(I) collagen in kidneys of uremic rats,” Kidney Int Suppl, vol. 62, 1997, pp. S15-S22.
15. M. Motojima, A. Hosokawa, H. Yamato, et al. “Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-kB and free radical in proximal tubular cells,” Kidney Int, vol. 63, no. 5, 2003, pp. 1671-1680.
16. D. Stavrou,” Neovascularisation in wound healing,” J Wound Care, vol, 17, no. 7, 2008, pp. 298-300.
17. P. Carmeliet, “Angiogenesis in health and disease,” Nat Med, vol. 9, no. 6, 2003, pp. 653-660.
18. H. Yamamoto, S. Tsuruoka, T. Ioka, et al. ”Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells,” Kidney Int, vol. 69, no. 10, 2006, pp. 1780–1785.
19. Z. Tumur and T. Niwa, “Indoxyl sulfate inhibits nitric oxide production and cell viability by inducing oxidative stress in vascular endothelial cells,” Am J Nephrol, vol. 29, no. 6, 2009, pp. 551 -557.
20. F. C. Barreto, D. V. Barreto, S. Liabeuf, et al. “Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients,” Clin J Am Soc Nephrol, vol. 4, no. 10, 2009, pp. 1551–1558.
21. K. Taki, Y. Tsuruta, T. “Niwa. Indoxyl sulfate and atherosclerotic risk factors in hemodialysis patients,” Am J Nephrol, vol. 27, no. 1, 2007, pp. 30-35.
22. R. Vanholder , R. De Smet , M. A. Waterloos, et al. “Mechanisms of uremic inhibition of phagocyte reactive species production: characterization of the role of p-cresol,” Kidney Int, vol. 47, no. 2, 1995, pp. 510-517
23. M. L. Wratten , C. Tetta , R. De Smet, et al. ”Uremic ultrafiltrate inhibits platelet-activating factor synthesis,” Blood Purif, vol. 17, no. 2-3, 1999, pp. 134-141.
24. A. W. Martinez , N. S. Recht , T. H. Hostetter, et al. “Removal of P-cresol sulfate by hemodialysis,” J Am Soc Nephrol, vol. 16, no. 11, 2005, pp. 3430-3436.
25. H. De Loor, B. Bammens, P. Evenepoel, et al. “ Gas chromatographic - mass spectrometric analysis for measurement of p-cresol and its conjugated metabolites in uremic and normal serum,” Clin Chem, vol. 51, no. 8, 2005, pp. 1535–1538.
26. E. Schepers, N. Meert , G. Glorieux, et al. “P-cresylsulphate, the main in vivo metabolite of p-cresol, activates leucocyte free radical production,” Nephrol Dial Transplant, vol. 22, no. 2, 2007, pp. 592-596.
27. J. P. Kirton and Q. Xu, “ Endothelial precursors in vascular repair,” Microvascular Research, vol. 79, no. 3, 2010, pp. 193-199.
28. N. Werner , S. Wassmann, P. Ahlers, et al. “Endothelial progenitor cells correlate with endothelial function in patients with coronary artery disease,” Basic Res Cardiol, vol. 102, no. 6, 2007, pp. 565–571.
29. B. A. Boilson, T. J. Kiernan, A. Harbuzariu, et al. “Circulating CD34+ cell subsets in patients with coronary endothelial dysfunction,” Nat. Clin. Pract. Cardiovasc. Med, vol. 5, no. 8, 2008, pp. 489–496.
30. R. N. Foley, P. S. Parfrey, M. J. Sarnak. et al. ” Epidemiology of cardiovascular disease in chronic renal disease,” J Am Soc Nephrol, vol. 9, no. Suppl 12, 1998, pp. S16–23.
31. G. A. Block, T. E. Hulbert-Shearon, N. W. Levin, et al. “Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study,” Am J Kidney Dis, vol. 31, no. 4, 1998, pp. 607-617.
32. C. Tomiyama, A. Higa, M. A. Dalboni, et al. ”The impact of traditional and non-traditional risk factors on coronary calcification in pre-dialysis patients,” Nephrol Dial Transplant, vol. 21, no. 9, 2006, pp. 2464-2471.
33. B. K. Meijers , S. Van Kerckhoven , K. Verbeke , et al. “The Uremic Retention Solute p-Cresyl Sulfate and Markers of Endothelial Damage,” Am J Kidney Dis, vol. 54, no. 5, 2009, pp. 891-901.
34. L. Dou, E. Bertrand, C. Cerini, et al. ”The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair,” Kidney Int, vol. 65, no. 2, 2004, pp. 442-451.
35. A. K. Gelasco and J. R. Raymond, ”Indoxyl sulfate induces complex redox alterations in mesangial cells,” Am J Physiol Renal Physiol, vol. 290, no. 5, 2006, pp. F1551-1558.
36. S. Rajagopalan, S. Dellegrottaglie, AL Furniss, et al. “Peripheral Arterial Disease in Patients With End-Stage Renal Disease , Observations From the Dialysis Outcomes and Practice Patterns Study (DOPPS),” Circulation, vol. 114, no. 18, 2006, pp. 1914-1922.
37. K. D. Gibson, M. T. Caps, T. R. Kohler, et al. “Assessment of a policy to reduce placement of prosthetic hemodialysis access,” Kidney Int. vol. 59, no. 6, 2001, pp. 2335-2345..
38. S. C. Chen , J. M. Chang , S. J. Hwang , et al. “Significant correlation between ankle-brachial index and vascular access failure in hemodialysis patients,” Clin J Am Soc Nephrol, vol. 4, no. 1, 2009, pp. 128-134.
39. L. Hofstra, J. H. Tordoir, P. J. Kitslaar, et al. “Enhanced cellular proliferation in intact stenotic lesions derived from human arteriovenous fistulas and peripheral bypass grafts. Does it correlate with flow parameters?,” Circulation, vol. 94, no. 6, 1996, pp. 1283-90.
40. R. Ross, ”The pathogenesis of atherosclerosis: A perspective for the 1990s,” Nature, vol. 362, no. 6423, 1993, pp. 801-809.
41. C. J. Lin, H. H. Chen, C. F. Pan, et al. “p-Cresylsulfate and indoxyl sulfate level at different stages of chronic kidney disease,” J Clin Lab Anal, vol. 25, no. 3, 2011, pp. 191-197.
42. D. H. Krieter , A. Hackl , A. Rodriguez, et al. “Protein-bound uraemic toxin removal in haemodialysis and post-dilution haemodiafiltration,” Nephrol Dial Transplant, vol. 25, no. 1, 2010, pp. 212-218.
43. C. J. Lin, H. L. Liu, C. F. Pan, et al. “Indoxyl sulfate predicts cardiovascular disease and renal function deterioration in advanced chronic kidney disease,” Archive of Medical Research, vol. 43, no. 6, 2012, pp. 451-456.
44. C. J. Lin, C. F. Pan , H. L. Liu, et al. “The role of protein-bound uremic toxins on peripheral artery disease and vascular access failure in patients on hemodialysis,” Atherosclerosis, vol. 225, no. 1, 2012, pp. 173-179.
45. C. J. Lin, H. L. Liu, C. F. Pan, et al. “P-cresyl sulfate predict cardiovascular and mortality in elderly hemodialysis patients,” Archive of medical Science, vol. 9, no. 4, 2013, pp. 662-668.
46. C. J. Lin, C. F. Pan, C. K. Chuang, et al. “GI related Uremic Toxins in Peritoneal Dialysis, A pilot Study with a 5-year follow-up,” Archive of Medical Research, vol. 44, no. 7, 2013, pp. 535-541.
47. C. J. Lin, C. F. Pan, C. K. Chuang, et al. “P-cresyl sulfate is a valuable predictor of clinical outcomes in Pre-ESRD patients,” Biomedical Research International, 2014, in press.
48. A. Ichihara, M. Hayashi, M. Ryuzaki, et al. ”Fluvastatin prevents development of arterial stiffness in hemodialysis patients with type 2 diabetes mellitus,” Nephrol Dial Transplant, vol. 17, no. 8, 2002, pp. 1513-1517.
49. M. K. Sigrist, M. W. Taal, P. Bungay, et al. “Progressive vascular calcification over 2 years is associated with arterial stiffening and increased mortality in patients with stages 4 and 5 chronic kidney disease,” Clin J Am Soc Nephrol, vol. 2, no. 6, 2007, pp. 1241-1248.
50. N. D. Toussaint, K. K. Lau, B. J. Strauss, et al. “Association between vascular calcification, arterial stiffness and bone mineral density in chronic kidney disease,” Nephrol Dial Transplant, vol. 23, no. 2, 2008, pp. 586-593.
51. A. Ichihara, M. Hayashi, Y. Kaneshiro, et al. “Low doses of losartan and trandolapril improve arterial stiffness in hemodialysis patients,” Am J Kidney Dis, vol. 45, no. 5, 2005, pp. 866-874.
52. T. Kitahara, K. Ono, A. Tsuchida, et al. “Impact of brachial-ankle pulse wave velocity and ankle-brachial blood pressure index on mortality in hemodialysis patients,” Am J Kidney Dis, vol. 46, no. 4, 2005, pp. 688-696.
53. S. Liabeuf, D. V. Barreto, F. C. Barreto, et al. “Free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease,” Nephrol Dial Transplant, vol. 25, no. 4, 2010, pp. 1183-1191.
54. F. Cofan , E. Vela , M. Clèries , et al. “Analysis of dyslipidemia in patients on chronic hemodialysis in Catalonia,” Atherosclerosis, vol. 184, no. 1, 2006, pp. 94-102.
55. D. Puskar, J. Pasini, I. Savic, et al. “Survival of primary arteriovenous fistula in 463 patients on chronic hemodialysis,” Croat Med J, vol. 43, no. 3, 2002, pp. 306-311.
論文全文使用權限:同意授權於2015-07-04起公開